We have developed a sensitive method for the detection and analysis of Gram-negative bacterial lipopolysaccharides (LPS) including meningococcal LOS on membranes. Specificity and sensitivity in the detection of LPSs blotted on nylon and polyvinylidene difluoride (PVDF) membranes were examined. An LPS or a mixture of protein markers was subjected to SDS-PAGE and separated into a series of LPS components with different molecular size or into individual proteins. The separated LPS components or proteins were then electrophoretically transferred onto the membranes. The LPS and proteins on the membranes were oxidized with periodate and reacted with a hydrazide containing steroid, digoxigenin. The LPS were visualized by alkaline phosphatase conjugated antibody against the steroid and the enzyme substrate, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. Both nylon and PVDF membranes provided high sensitivity in the detection of LPSs. However, the PVDF membrane had nonspecific staining of proteins. The banding patterns of both smooth and rough LPSs from different bacterial species were similar to those of silver stained gels. The sensitivity of detection for Pseudomonas aeruginosa smooth LPS (F-D type 1) or meningococcal LOS was 10-20 ng. This chemical detection in combination with antibody or lectin detection of an LPS blotted on the same piece of membrane provides a precise way to determine the reactive component in a multi-component LPS.
